DESCRIPTION: Recently, legalization of marijuana use has been a topic of significant public attention, yet our understanding of the fundamental underlying mechanisms remains rudimentary. ?9-tetrahydrocannabinol (THC), the main psychoactive ingredient in marijuana, can interact with the endogenous cannabinoid system (eCBs), which includes cannabinoid receptors, endogenous ligands, and enzymes involved in the synthesis and metabolism of these ligands. The finding that developmental cannabis exposure in humans is associated with psychiatric vulnerability and addiction later in life suggests that eCBs may influence brain development and plasticity in significant ways. However, how eCBs exert such influences remains poorly understood. This is in large part due to the broad expression of eCBs both spatially and temporally, making constitutive knockout phenotypes difficult to interpret, due to developmental compensation and lack of specificity. Thus, it requires new tools and technologies to elucidate cell type-specific and time-dependent functions of eCBs. The zebrafish Danio rerio is a vertebrate model organism well suited to connect brain development with function. The transparent and easily accessible embryos and larvae offer the opportunity to observe brain development at single-cell levels. As early as 5 days post fertilization (dpf), larva zebrafish need to hunt for food and escape from predators, thus a simple yet functional reward system is in place. Embryonic and larval zebrafish can be conveniently exposed to chemicals (e.g. drugs of abuse) for studying their biological effects. The ability to control gene activity ith spatiotemporal precision shall greatly facilitate new discoveries in biology and medicine. The recent development of a novel RNA-guided genome-editing technology named CRISPR offers unprecedented ease for studying gene function in vivo. In this exploratory R21 proposal, we will adapt the in vitro CRISPR gene regulation technologies for spatiotemporally controllable gene silencing and activation in neurons in vivo using light, which we term Opto-CRISPR. The specific hypothesis that we will test with this new technology is: the cannabinoid signaling regulates the development of dopaminergic (DA) neurons related to reward circuitry. Impact: If successful, this project will exert a major impact on the following research fields: 1) It will advance technologies for spatiotemporally targetable gene silencing and activation in neurons in vivo. We will make this technology widely available to the research community. 2) It will reveal whether eCBs regulate the development of DA neurons related to reward circuitry. Such new knowledge will help inform policy decisions and foster innovations for treating drug abuse and addiction. Suitability for CEBRA: (1) This project will test a highly novel and significant hypothesis, aimed at evaluating the role of eCBs in the development of DA neurons related to reward circuitry. At the moment, there are scant precedents or preliminary data regarding this topic, however, if confirmed, it would have a substantial impact on current thinking of eCBs signaling in the reward circuit development. (2) This project will develop innovative techniques for gene silencing and activation with high spatiotemporal precision. This will have promising applicability to drug abuse research and beyond.